Isolation of the side population from neurogenic niches enriches for endothelial cells

In stem cell research, DNA-binding dyes offer the ability to purify live stem cells using flow cytometry as they form a low-fluorescence side population due to the activity of ABC transporters. Adult neural stem cells exist within the lateral ventricle and dentate gyrus of the adult brain yet the ability of DNA-binding dyes to identify these adult stem cells as side populations remains untested. The following experiments utilize the efflux of a DNA-binding dye, Vyrbant DyeCycle Violet (DCV), to isolate bona fide side populations in the mouse dentate gyrus and subventricular zone (SVZ), and test their sensitivity to ABC transporter inhibitors. A distinct side population was found in both the adult lateral ventricle and dentate gyrus using DCV fluorescence and forward scatter instead of the conventional dual fluorescence approach. These side populations responded strongly to inhibition with the ABC transporter antagonists, verapamil and fumitremorgin C. The majority of the cells residing in the side populations of dentate gyrus and SVZ were characterized by their expression of CD31. Additionally, at least 90% of all CD31+ cells found in the dentate gyrus and SVZ were negative for the hematopoietic marker CD45, leading to the hypothesis that the CD31+ cells in the side population were endothelial cells. These findings, therefore, suggest that the side population analysis provides an efficient method to purify CD31-expressing endothelial cells, but not adult neural stem cells.